臺灣博碩士論文加值系統

木材對植物的生長發育扮演重要角色，同時也是人類生活中不可或缺的生物資源。而木材形成是相當複雜的生長發育過程，至今其調控機制所知甚少，且多為單一物種中與少數轉錄因子之研究。因此為系統性地探討木材形成的轉錄調控機制，實驗室先前的研究以木本模式植物毛果楊(Populus trichocarpa)為對象，利用轉錄組測序分析(RNA-sequencing)篩選出182個木質部專一表達之轉錄因子，再加上4個以酵母菌單雜合系統(yeast one-hybrid assay)分析得知會結合木質素生合成基因啟動子的轉錄因子，欲透過DNA親合純化測序分析(DNA affinity purification sequencing)建立木材形成之基因調控網絡(gene regulatory network)。然而該網絡僅能探討轉錄因子能否結合下游基因，無法得知轉錄因子如何進行調控。因此本研究將轉錄因子對毛果楊木質部原生質體進行轉殖以過量表達，並抽取total RNA進行轉錄組測序、篩選差異表達基因，該實驗方法稱為原生質體轉錄體活性分析(protoplast transactivation assay-RNA sequencing)。本研究建立毛果楊木質部原生質體轉錄體活性分析系統，得以大規模對基因調控網絡進行功能性驗證，與DNA親合純化測序分析之結果進行合併便可建構出更加完整的木材形成基因調控網絡。本研究進而對玫瑰桉(Eucalyptus grandis)與中國鵝掌楸(Liriodendron chinense)利用相似策略進行探討，以研究木材形成的調控機制在不同物種中是否存在保守性。透過建立毛果楊、玫瑰桉與中國鵝掌楸轉錄因子家族的親緣關係樹，可得知有若干木質部專一表達的轉錄因子有群聚之現象，顯示這些轉錄因子可能具有相似的調控功能。最後，本研究篩選玫瑰桉中13個木質部專一表達的轉錄因子，以進行往後DNA親合純化測序分析的實驗。未來期望透過比較多個物種之木材形成基因調控網絡，能夠較全面且系統性地探究木材形成的保守轉錄調控機制，以進一步了解木本植物的生長發育過程，並協助木材的育種以及品質改良。

Wood plays an essential role in plant growth, and it is also a valuable bio-based material. Wood formation is a complicated developmental process, but the entire regulatory mechanism is still elusive. Previous studies related to wood formation usually focused on few transcription factor (TF) in single species, thus lacking a comprehensive view of how wood formation is regulated. To obtain more comprehensive knowledge, 186 TFs from Populus trichocarpa were chosen to construct a gene regulatory network (GRN) using DNA affinity purification sequencing (DAP-seq) in our laboratory. Among 186 TFs, 182 TFs were selected as xylem specific TFs by RNA-sequencing, and 4 TFs which physically bind to promoters of lignin biosynthesis genes were selected by yeast one-hybrid assay. However, the network only shows the physical interaction between the TFs and their downstream targets. In this study, the TFs of interested were transiently over-expressed in P. trichocarpa xylem protoplasts. The total RNA was isolated, and carried on to RNA-sequencing. The method was called protoplast transactivation assay-RNA sequencing (PTA-seq). PTA-seq can help us accomplish TF functional characterization in a large scale. Therefore, the combination of PTA-seq and DAP-seq provides a more precise insight in studying wood formation. Similar pipeline was also used to investigate conserved wood formation regulatory mechanism in Eucalyptus grandis and Liriodendron chinense. The phylogenetic trees indicated that several xylem specific TFs are conserved in P. trichocarpa, E. grandis, and L. chinense. Finally, 13 xylem specific TFs from E. grandis were selected for further DAP-seq investigation. We aim to discover the conserved wood formation transcriptional regulatory mechanism in a systematic way, to further support forest tree breeding and wood property improvement.

中文摘要------------------------------------------------------------------------------------ p.1
英文摘要(Abstract)------------------------------------------------------------------------ p.2
目錄------------------------------------------------------------------------------------------ p.3-4
圖目錄--------------------------------------------------------------------------------------- p.5-8
表目錄--------------------------------------------------------------------------------------- p.9
一、前言------------------------------------------------------------------------------------ p.10-15
二、材料方法------------------------------------------------------------------------------ p.16-24
(一) 實驗材料之培養與採集----------------------------------------------------- p.16
(二) 建構毛果楊木材形成基因調控網路所使用的轉錄因子之選擇----- p.16-17
(三) 基因選殖相關技術與載體建構-------------------------------------------- p.17
(四) 毛果楊木質部原生質體轉錄體活性分析-------------------------------- p.17-22
(五) 玫瑰桉與中國鵝掌楸total RNA之提取---------------------------------- p.22-23
(六) 毛果楊、玫瑰桉與中國鵝掌楸轉錄因子家族親緣關係樹之建立---- p.23-24
三、實驗結果------------------------------------------------------------------------------- p.25-37
Part 1. 建立毛果楊木質部原生質體轉錄體活性分析系統----------------------- p.25-31
(一) 毛果楊木質部原生質體轉錄體活性分析之實驗流程----------------- p.25-26
(二) 氯化銫—溴化乙錠超高速梯度離心的純化載體DNA---------------- p.26
(三) 毛果楊木質部原生質體小量轉殖與轉殖效率的計算----------------- p.26
(四) 毛果楊木質部原生質體total RNA完整度(RIN)之提升--------------- p.27-31
Part 2. 利用木質部原生質體轉錄體活性分析與DNA親和純化測序分析建立小規
模之毛果楊木材形成基因調控網絡--------------------------------------------- p.31-33
(一) 毛果楊木質部原生質體轉錄體活性分析與DNA親和純化測序分析結果之合併-------------------------------------------------------------------------- p.31-33
(二) 與前人研究比較實驗結果之再現性，以PtrSND1-B1為例------------ p.33
Part 3. 木材形成基因調控機制在跨物種間保守性的探討----------------------- p.33-37
(一) 跨物種研究對象之選擇----------------------------------------------------- p.33-34
(二) 玫瑰桉與中國鵝掌楸木質部專一表達轉錄因子之分析-------------- p.34
(三) 毛果楊、玫瑰桉與中國鵝掌楸不同轉錄因子家族親緣關係樹之建立---- -------------------------------------------------------------------------------------------p.34-37
(四) 玫瑰按轉錄因子DNA親和純化測序分析之載體建構---------------- p.37
四、討論------------------------------------------------------------------------------------- p.38-42
(一) 本論文所建立之毛果楊木質部原生質體轉錄體活性分析系統可大規模對基因調控網絡進行功能性驗證------------------------------------------------------- p.38
(二) 美國、哈爾濱、台灣三地毛果楊木質部原生質體RNA完整度(RIN)之差
異--------------------------------------------------------------------------------------- p.38-39
(三) WI solution中甘露醇濃度對毛果楊木質部原生質體RNA完整度之可能影
響--------------------------------------------------------------------------------------- p.39-40
(四) 以毛果楊木質部原生質體轉錄體活性分析與DNA親和純化測序分析建立毛果楊木材形成基因調控網絡之未來展望--------------------------------- p.40-41
(五) 探究木材形成基因調控機制於不同物種間保守性之未來展望----- p.41-42
五、參考文獻------------------------------------------------------------------------------- p.43-47
六、圖附錄--------------------------------------------------------------------------------- p.48-101
七、表附錄------------------------------------------------------------------------------- p.102-105

Bartlett, A., O'Malley, R.C., Huang, S.C., Galli, M., Nery, J.R., Gallavotti, A., and Ecker, J.R. (2017). Mapping genome-wide transcription-factor binding sites using DAP-seq. Nature Protocols 12, 1659-1672.
Birnbaum, K., Shasha, D.E., Wang, J.Y., Jung, J.W., Lambert, G.M., Galbraith, D.W., and Benfey, P.N. (2003). A Gene Expression Map of the Arabidopsis Root. Science 302, 1956-1960.
Bose, B.K. (2010). Global Warming: Energy, Environmental Pollution, and the Impact of Power Electronics. IEEE Industrial Electronics Magazine 4, 6-17.
Brown, K., Takawira, L.T., O'Neill, M.M., Mizrachi, E., Myburg, A.A., and Hussey, S.G. (2019). Identification and functional evaluation of accessible chromatin associated with wood formation in Eucalyptus grandis. New Phytologist 223, 1937-1951.
Chai, G., Wang, Z., Tang, X., Yu, L., Qi, G., Wang, D., Yan, X., Kong, Y., and Zhou, G. (2014). R2R3-MYB gene pairs in Populus: evolution and contribution to secondary wall formation and flowering time. Journal of Experimental Botany 65, 4255-4269.
Chen, H., Wang, J.P., Liu, H., Li, H., Lin, Y.-C.J., Shi, R., Yang, C., Gao, J., Zhou, C., Li, Q., Sederoff, R.R., Li, W., and Chiang, V.L. (2019a). Hierarchical Transcription Factor and Chromatin Binding Network for Wood Formation in Populus trichocarpa. Plant Cell 31, 602-626.
Chen, J., Hao, Z., Guang, X., Zhao, C., Wang, P., Xue, L., Zhu, Q., Yang, L., Sheng, Y., Zhou, Y., Xu, H., Xie, H., Long, X., Zhang, J., Wang, Z., Shi, M., Lu, Y., Liu, S., Guan, L., Zhu, Q., Yang, L., Ge, S., Cheng, T., Laux, T., Gao, Q., Peng, Y., Liu, N., Yang, S., and Shi, J. (2019b). Liriodendron genome sheds light on angiosperm phylogeny and species–pair differentiation. Nature Plants 5, 18-25.
Du, J., and Groover, A. (2010). Transcriptional Regulation of Secondary Growth and Wood Formation. Journal of Integrative Plant Biology 52, 17-27.
Groover, A.T. (2005). What genes make a tree a tree? Trends in Plant Science 10, 210-214.
Gui, J., Luo, L., Zhong, Y., Sun, J., Umezawa, T., and Li, L. (2019). Phosphorylation of LTF1, an MYB Transcription Factor in Populus, Acts as a Sensory Switch Regulating Lignin Biosynthesis in Wood Cells. Molecular Plant 12, 1325-1337.
Hens, K., Feuz, J.-D., Isakova, A., Iagovitina, A., Massouras, A., Bryois, J., Callaerts, P., Celniker, S.E., and Deplancke, B. (2011). Automated protein-DNA interaction screening of Drosophila regulatory elements. Nature Methods 8, 1065-1070.
Hinchee, M., Rottmann, W., Mullinax, L., Zhang, C., Chang, S., Cunningham, M., Pearson, L., and Nehra, N. (2011). Short-Rotation Woody Crops for Bioenergy and Biofuels Applications. In Biofuels: Global Impact on Renewable Energy, Production Agriculture, and Technological Advancements, D. Tomes, P. Lakshmanan, and D. Songstad, eds (New York, NY: Springer New York), pp. 139-156.
Jiao, B., Zhao, X., Lu, W., Guo, L., and Luo, K. (2019). The R2R3 MYB transcription factor MYB189 negatively regulates secondary cell wall biosynthesis in Populus. Tree Physiology 39, 1187-1200.
Kirst, M., Johnson, A.F., Baucom, C., Ulrich, E., Hubbard, K., Staggs, R., Paule, C., Retzel, E., Whetten, R., and Sederoff, R. (2003). Apparent homology of expressed genes from wood-forming tissues of loblolly pine (Pinus taeda) with Arabidopsis thaliana. Proceedings of the National Academy of Sciences 100, 7383.
Li, C., Wang, X., Ran, L., Tian, Q., Fan, D., and Luo, K. (2015). PtoMYB92 is a Transcriptional Activator of the Lignin Biosynthetic Pathway During Secondary Cell Wall Formation in Populus tomentosa. Plant and Cell Physiology 56, 2436-2446.
Li, C., Wang, X., Lu, W., Liu, R., Tian, Q., Sun, Y., and Luo, K. (2014). A poplar R2R3-MYB transcription factor, PtrMYB152, is involved in regulation of lignin biosynthesis during secondary cell wall formation. Plant Cell, Tissue and Organ Culture 119, 553-563.
Li, E., Bhargava, A., Qiang, W., Friedmann, M.C., Forneris, N., Savidge, R.A., Johnson, L.A., Mansfield, S.D., Ellis, B.E., and Douglas, C.J. (2012a). The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus. New Phytologist 194, 102-115.
Li, Q., Lin, Y.-C., Sun, Y.-H., Song, J., Chen, H., Zhang, X.-H., Sederoff, R.R., and Chiang, V.L. (2012b). Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa. Proceedings of the National Academy of Sciences 109, 14699-14704.
Li, S., Lin, Y.-C.J., Wang, P., Zhang, B., Li, M., Chen, S., Shi, R., Tunlaya-Anukit, S., Liu, X., Wang, Z., Dai, X., Yu, J., Zhou, C., Liu, B., Wang, J.P., Chiang, V.L., and Li, W. (2019). The AREB1 Transcription Factor Influences Histone Acetylation to Regulate Drought Responses and Tolerance in Populus trichocarpa. Plant Cell 31, 663-686.
Lin, Y.-C., Li, W., Sun, Y.-H., Kumari, S., Wei, H., Li, Q., Tunlaya-Anukit, S., Sederoff, R.R., and Chiang, V.L. (2013). SND1 Transcription Factor–Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa. Plant Cell 25, 4324-4341.
Lin, Y.-C., Li, W., Chen, H., Li, Q., Sun, Y.-H., Shi, R., Lin, C.-Y., Wang, J.P., Chen, H.-C., Chuang, L., Qu, G.-Z., Sederoff, R.R., and Chiang, V.L. (2014). A simple improved-throughput xylem protoplast system for studying wood formation. Nature Protocols 9, 2194-2205.
Lin, Y.-C.J., Chen, H., Li, Q., Li, W., Wang, J.P., Shi, R., Tunlaya-Anukit, S., Shuai, P., Wang, Z., Ma, H., Li, H., Sun, Y.-H., Sederoff, R.R., and Chiang, V.L. (2017). Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa. Proceedings of the National Academy of Sciences 114, E9722-E9729.
Lin, Y.-H., Lu, Y.-C., Hsu, J.S., Lee, K.-H., Cheng, Y.-W., Chen, Y.-C., Chen, T.-F., Tu, C.-T., Hsu, C.-M., Chou, C.-C., Chen, P.-L., Tu, Y.-C.E., and Chen, C.-Y. (2019). variant2literature: full text literature search for genetic variants. bioRxiv, 583450.
Lu, S., Li, Q., Wei, H., Chang, M.-J., Tunlaya-Anukit, S., Kim, H., Liu, J., Song, J., Sun, Y.-H., Yuan, L., Yeh, T.-F., Peszlen, I., Ralph, J., Sederoff, R.R., and Chiang, V.L. (2013). Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa. Proceedings of the National Academy of Sciences 110, 10848-10853.
Martin, L., Decourteix, M., Badel, E., Huguet, S., Moulia, B., Julien, J.-L., and Leblanc-Fournier, N. (2014). The zinc finger protein PtaZFP2 negatively controls stem growth and gene expression responsiveness to external mechanical loads in poplar. New Phytologist 203, 168-181.
McCarthy, R.L., Zhong, R., Fowler, S., Lyskowski, D., Piyasena, H., Carleton, K., Spicer, C., and Ye, Z.-H. (2010). The Poplar MYB Transcription Factors, PtrMYB3 and PtrMYB20, are Involved in the Regulation of Secondary Wall Biosynthesis. Plant and Cell Physiology 51, 1084-1090.
Myburg, A.A., Grattapaglia, D., Tuskan, G.A., Hellsten, U., Hayes, R.D., Grimwood, J., Jenkins, J., Lindquist, E., Tice, H., Bauer, D., et al. (2014). The genome of Eucalyptus grandis. Nature 510, 356-362.
Noh, S.A., Choi, Y.-I., Cho, J.-S., and Lee, H. (2015). The poplar basic helix-loop-helix transcription factor BEE3 – Like gene affects biomass production by enhancing proliferation of xylem cells in poplar. Biochemical and Biophysical Research Communications 462, 64-70.
Quesada, T., Li, Z., Dervinis, C., Li, Y., Bocock, P.N., Tuskan, G.A., Casella, G., Davis, J.M., and Kirst, M. (2008). Comparative analysis of the transcriptomes of Populus trichocarpa and Arabidopsis thaliana suggests extensive evolution of gene expression regulation in angiosperms. New Phytologist 180, 408-420.
Smit, M.E., McGregor, S.R., Sun, H., Gough, C., Bågman, A.-M., Soyars, C.L., Kroon, J.T., Gaudinier, A., Williams, C.J., Yang, X., Nimchuk, Z.L., Weijers, D., Turner, S.R., Brady, S.M., and Etchells, J.P. (2020). A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis. Plant Cell 32, 319-335.
Takata, N., Awano, T., Nakata, M.T., Sano, Y., Sakamoto, S., Mitsuda, N., and Taniguchi, T. (2019). Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells. Tree Physiology 39, 514-525.
Tang, X., Zhuang, Y., Qi, G., Wang, D., Liu, H., Wang, K., Chai, G., and Zhou, G. (2015). Poplar PdMYB221 is involved in the direct and indirect regulation of secondary wall biosynthesis during wood formation. Scientific Reports 5, 12240.
Tian, Q., Wang, X., Li, C., Lu, W., Yang, L., Jiang, Y., and Luo, K. (2013). Functional characterization of the poplar R2R3-MYB transcription factor PtoMYB216 involved in the regulation of lignin biosynthesis during wood formation. PLoS one 8, e76369-e76369.
Tuskan, G.A., DiFazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U., Putnam, N., Ralph, S., Rombauts, S., Salamov, A., Schein, J., et al. (2006). The Genome of Black Cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596-1604.
Wang, J., Andersson-Gunnerås, S., Gaboreanu, I., Hertzberg, M., Tucker, M.R., Zheng, B., Leśniewska, J., Mellerowicz, E.J., Laux, T., Sandberg, G., and Jones, B. (2011). Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis. PLoS one 6, e28878-e28878.
Wang, W., Li, E., Porth, I., Chen, J.-G., Mansfield, S.D., Douglas, C.J., and Wang, S. (2016). Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis. Journal of Plant Biology 59, 16-23.
Xu, C., Fu, X., Liu, R., Guo, L., Ran, L., Li, C., Tian, Q., Jiao, B., Wang, B., and Luo, K. (2017). PtoMYB170 positively regulates lignin deposition during wood formation in poplar and confers drought tolerance in transgenic Arabidopsis. Tree Physiology 37, 1713-1726.
Xu, C., Shen, Y., He, F., Fu, X., Yu, H., Lu, W., Li, Y., Li, C., Fan, D., Wang, H.C., and Luo, K. (2019). Auxin-mediated Aux/IAA-ARF-HB signaling cascade regulates secondary xylem development in Populus. New Phytologist 222, 752-767.
Yang, L., Zhao, X., Yang, F., Fan, D., Jiang, Y., and Luo, K. (2016). PtrWRKY19, a novel WRKY transcription factor, contributes to the regulation of pith secondary wall formation in Populus trichocarpa. Scientific Reports 6, 18643.
Yang, L., Zhao, X., Ran, L., Li, C., Fan, D., and Luo, K. (2017). PtoMYB156 is involved in negative regulation of phenylpropanoid metabolism and secondary cell wall biosynthesis during wood formation in poplar. Scientific Reports 7, 41209.
Ye, Z.-H., and Zhong, R. (2015). Molecular control of wood formation in trees. Journal of Experimental Botany 66, 4119-4131.
Yordanov, Y.S., Regan, S., and Busov, V. (2010). Members of the LATERAL ORGAN BOUNDARIES DOMAIN Transcription Factor Family Are Involved in the Regulation of Secondary Growth in Populus. Plant Cell 22, 3662-3677.
Zhong, R., Demura, T., and Ye, Z.-H. (2006). SND1, a NAC Domain Transcription Factor, Is a Key Regulator of Secondary Wall Synthesis in Fibers of Arabidopsis. Plant Cell 18, 3158.
Zhong, R., Lee, C., and Ye, Z.-H. (2010a). Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors. Plant Physiology 152, 1044-1055.
Zhong, R., Lee, C., and Ye, Z.-H. (2010b). Evolutionary conservation of the transcriptional network regulating secondary cell wall biosynthesis. Trends in Plant Science 15, 625-632.
Zhong, R., McCarthy, R.L., Lee, C., and Ye, Z.-H. (2011). Dissection of the Transcriptional Program Regulating Secondary Wall Biosynthesis during Wood Formation in Poplar. Plant Physiology 157, 1452-1468.
Zhong, R., McCarthy, R.L., Haghighat, M., and Ye, Z.-H. (2013). The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation. PLoS one 8, e69219-e69219.